Hepatitis C virus (HCV) is a major health problem and the leading cause of chronic liver disease throughout the world. It is estimated that at least 180 million people worldwide are chronically infected with HCV. In Vietnam, the proportion of infected HCV individuals in the population is 4-9%. Approximately 55-85% of acutely infected HCV individuals will convert to chronic infection, 5-25% of these chronic carriers are at risk of developing cirrhosis after 25-30 years and of those with cirrhosis, 30% are at risk of liver decompensation over 10 years, and 1-3% will develop liver cancer each year. According to epidemiological research, HCV is the cause of 40% of individuals in final stage cirrhosis and 60% in hepatoma.
Currently, α-interferons (AI) are the therapies of choice for the treatment of chronic HCV infection. AI can give a persistent response to HCV in approximately 70% of cases, however these interferons cause many side-effects, even in the case of PEG-interferon alpha. These side-effects can sometimes limit treatment, leaving the treatment of patients incomplete. Side-effects include influenza-like symptoms and hematologic effects such as thalassemia and anemia.
Interferons are currently used for the treatment of many viral diseases such as hepatitis B, hepatitis C, hepatitis D, condyloma acuminata, lepromatous leprosy, chronic leukaemia and AIDS. AI are also effective in reducing malignant tumors and treating Kaposi's sarcoma, melanoma, and renal cell carcinoma. Moreover, AI are applicable in the prevention and treatment of diseases in cattle and other livestock. For example, AI enhance the activity of vaccines used in prophylaxis and treatment of foot and mouth disease and porcine reproductive and respiratory syndrome.
AI have been produced from human cell lines incubated in tissue culture media or leukocytes derived from donors. However, these methods are time consuming, labor intensive, expensive, and not amenable to large scale manufacturing. Furthermore, there is the risk of septicaemia caused by infectious agents from the cell lines.
With the development of recombinant DNA technology, we can now introduce AI genes into microorganisms that enable production of large amounts of interferons. However, these methods also present certain advantages and difficulties, mainly in the steps of expression and large-scale protein production.
IL-29 is a member of the helical cytokine family and is a type III interferon. It is also known as interferon lambda 1 (IFNλ1) and is highly similar in amino acid sequence to IL-28, the other type III interferon. IL-28 and IL-29 (IFNλ1) were recently described as members of a new cytokine family that shares with type I interferon (IFN), the same Jak/Stat signaling pathway driving expression of a common set of genes. Accordingly, they have been named IFNλ. IFNsλ exhibit several common features with type I IFNs: antiviral activity, antiproliferative activity and in vivo antitumor activity. Importantly, however, IFNsλ bind to a distinct membrane receptor, composed of IFNLR1 and IL10R2.
The major disadvantage with the therapeutic use of most biologicals is that they are administered parenterally, e.g. intravenously (i.v.), subcutaneously (s.c.), intramuscularly (i.m.) etc. This means that delivery to the patient is associated with pain and discomfort. Furthermore, because of their usually very short half-lives, biologicals require frequent administration to the patient in order to maintain therapeutic blood serum or plasma levels of the drug. Injections that cannot be self-administered require frequent trips to the clinic and trained medical personnel, making such therapy inconvenient and expensive. Interferon alpha-2a (Roferon, Roche) and interferon alpha-2b (Intron A, Schering A G), the two recombinant forms of human interferon alpha used in the treatment of chronic hepatitis B and C, have a serum half-life of less than 12 h (McHutchison, et al., Engl. J. Med. 1998, 339, 1485-1492; Glue, et al., Clin. Pharmacol. Ther. 2000, 68, 556-567) and therefore require administration 3 times a week. Repeated injections with interferon beta-1b (Betaseron) are also required to treat the patients of multiple sclerosis (MS).
One very successful and well accepted method of overcoming the above requirement of frequent high dose injections to maintain threshold levels of the drug in the body is to increase the in vivo half-life of the therapeutic protein by conjugating it with a polymer, such as polyethylene glycol (PEG or Peg). PEG molecules with their long chains not only create a protective shield around the pegylated drug molecule in aqueous solution, thereby reducing the immunogenicity of protein drugs while also protecting them from the action of proteases, but they further help increase the circulation half-life of the drug by increasing its hydrodynamic volume which reduces its loss from the filtration mechanisms of the kidney glomeruli network. After their separation from the protein molecule, the PEG moieties are cleared without any structural changes and their clearance is proportional to their molecular weight.
Usually PEG moieties are attached to the protein by first activating the PEG moiety and then reacting the activated PEG agent with the side chains of an amino acid of a protein, such as the lysine residue and/or the N-terminal amino group on the protein. The most frequently used PEG is monofunctional PEG because this moiety resists cross-linking and aggregation. One such example has been disclosed by Davis et al. in U.S. Pat. No. 4,179,337.